1. Field of the Invention
The invention is in the field of molecular biology as related to the control of programmed cell death.
2. Description of the Background Art
Programmed Cell Death
Apoptosis, also referred to as programmed cell death or regulated cell death, is a process by which organisms eliminate unwanted cells. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1950); Ellis et al., Dev. 112:591-603 (1991); Vaux et al., Cell 76:777-779 (1994)). Programmed cell death can also act to regulate cell number, to facilitate morphogenesis, to remove harmful or otherwise abnormal cells and to eliminate cells that have already performed their function. Additionally, programmed cell death is believed to occur in response to various physiological stresses such as hypoxia or ischemia. The morphological characteristics of apoptosis include plasma membrane blebbing, condensation of nucleoplasm and cytoplasm and degradation of chromosomal DNA at inter-nucleosomal intervals. (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34) and occurs when a cell activates its internally encoded suicide program as a result of either internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wylie, A. H., et al., Int. Rev. Cyt. 68: 251 (1980); Ellis, R. E., etal., Ann. Rev. Cell Bio. 7:663 (1991)). In many cases, gene expression appears to be required, since cell death can be prevented by inhibitors of RNA or protein synthesis (Cohen et al, J. Immunol. 32:38-42 (1984); Stanisic et al., Invest. Urol. 16:19-22 (1978); Martin et al., J. Cell Biol. 106:829-844 (1988). A genetic pathway of programmed cell death was first identified in the nematode C. elegans. In this worm, the products of ced-3 and ced-4 genes carry out the program of cellular suicide (Yuan and Horvitz, Dev. Bio. 138: 33 (1990)).
Interleukin-1xcex2 Converting Enzyme
The mammalian homologue of the ced-3 gene product is interleukin-1xcex2 converting enzyme (ICE), a cysteine protease responsible for the activation of interleukin-1xcex2 (IL-1xcex2) (Thomberry, N. A., etal., Nature 356: 768 (1992); Yuan, J., et al., Cell 75: 641 (1993); Miura, M., et al., Cell 75: 653 (1993)). The Ice gene is a member of a family of genes. The mammalian ICE/Ced-3 family now includes at least six members: ICE, ICH-1/NEDD2, CPP32/Yama/Apopain, TX/ICEreIII/ICH-2, ICEreIIII and MCH2 (Yuan et al., Cell 75:641-652 (1993); Wang et al., Cell 78:739-750 (1994); Kumar et al., Genes Dev. 8:1613-1626 (1994); Fernandes-Alnerrni et al., J. Biol. Chem. 269:30761-30764 (1994); Tewari, M., et al., Cell 81:801-809 (1995); Nicholson, D., et al., Nature 376:37-43 (1995); Faucheu, C., et al., J. Biol. Chem. 269:30761-30764 (1994); Munday, N. A., et al., J. Biol. Chem. 270:15870-15876 (1995); Kamens, J., et al., J. Biol. Chem. 270:15250-15256 (1995); Femandes-Alnermi, et al., Canc. Res 55:2737-2742 (1994)).
Interleukin-1xcex2 converting enzyme (ICE) is a substrate-specific cysteine protease that cleaves the inactive 31 KD prointerleukin-1xcex2 at Asp116-Ala117, releasing a carboxy-terminal 153 amino-acid peptide to produce the mature 17.5 kD interleukin-1xcex2 (IL1xcex2) (Kostura et al., Proc. Natl. Acad. Sci., USA 86:5227-5231 (1989); Black et al., FEBS Lett. 247:386-390 (1989); Cerretti et al., Science 256:97-100 (1992); Thomberry et al., Nature 356:768-774 (1992)). Since this is member of a family of proteases whose active site cysteine residue is essential for ICE-mediated apoptosis, their proteolytic activity appears critical in mediating cell death (Miura et al., J. Cell 75:653-660 (1993)). IL1xcex2 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias (Dinarello, C. A., Blood 77:1627-1652 (1991); diGiovine et al., Today 11:13 (1990)).
A specific inhibitor of ICE, the crmA gene product of cowpox virus, prevents the proteolytic activation of IL-1xcex2 (Ray et al., Cell 69:597-604 (1992)) and also inhibits host inflammatory response (Ray et al., Cell 69:597-604 (1992)). Cowpox virus carrying a deleted crmA, gene is unable to suppress the inflammatory response of chick embryos, resulting in a reduction in the number of virus-infected cells and less damage to the host (Palumbo et al., Virology 171:262-273 (1989)). This observation indicates the importance of ICE in bringing about the inflammatory response.
It has also been shown that ICE overexpression induces apoptosis, and that mature IL-1xcex2 is released during cell death (Miura, M., et al., Cell 75: 653 (1993); Miura, M., et al., Proc. Natl. Acad. Sci. USA. 92:8318-8322, (1995). The cowpox virus gene product CrmA, a member of the serpin family and an inhibitor of ICE also prevents apoptosis (Miura, M., et al., Cell 75: 653 (1993); Miura, M., et al., Proc. Natl. Acad. Sci. USA. (In press); Ray, C. A., et al., Cell 69: 597 (1992); Gagliardini, V., et al., Science 263: 826 (1993); Boudreau, N., et al., Science 267: 891 (1995); Enari, M., et al., Nature 375: 78 (1995); Los, M., et al., Nature 375: 81 (1995)). In addition, the ability of CrmA to inhibit apoptosis correlates with its ability to inhibit mature IL-1xcex2 production. Recent reports indicate that tumor necrosis factor-xcex1 TNF-xcex1) induced apoptosis is mediated through a CrmA-inhibitable pathway suggesting involvement of the ICE family (Tewary, M., et al., J. Biol. Chem 270: 3255 (1995); Hsu, H., et al., Cell 81: 495 (1995); Miura, M., et al., Natl. Acad Sci. U.S.A. (In press)).
While the critical role of the ICE family in cell death is well accepted, the function of mature IL-1xcex2 in apoptosis is controversial. IL-1xcex2 has been shown to induce apoptosis in some systems (Onozaki et al., Immun 135:3962-3968 (1985); Ankarcrona etal., Exp. Cell Res. 213:172-177 (1994); Fratelli, M., etal., Blood 85:3532-3637 (1995)), and to prevent it in others (Belizario and Dinarello, Cancer Res. 51:2379-2385 (1991); Strijbos and Rothwell, J. Neurosci. 15:3468-3474 (1995)). Mature IL-1xcex2 has not only been detected in the media of TNF-xcex1 treated apoptotic fibroblasts, but also in the media of macrophages undergoing apoptosis following Shigella flexneri infection (Zychlinsky, A., et al., J. Clin. Invest. 94: 1328 (1994)). The detection of mature IL-1xcex2 release during apoptosis provides strong evidence for ICE itself being activated in cell death, since in-vivo ICE is the major (if not the only) protease responsible for the processing of proIL-1xcex2 as demonstrated in ICE deficient mice (Li, P., et al., Cell 80: 401 (1995); (Kuida, K., et al., Science 267: 2000 (1995)).
Tumor Necrosis Factor
Tumor necrosis factor-xcex1 (TNF-xcex1) is a pleiotropic tumoricidal cytokine (Tracey, K. J. et al., Ann. Rev. Cell. Biol. 9:317-343 (1993)). One of the striking functions of TNF-xcex1 is to induce apoptosis of transformed cells. In the case of non-transformed cells, TNFxcex1 can also induce apoptosis in the presence of metabolic inhibitors (Tracey, K. J., et al., Ann. Rev. Cell. Biol. 9:317-343 (1993). Apoptosis induced by TNF-xcex1 is also suppressed by bcl-2.
One of the most extensively studied functions of TNF-xcex1 is its cytotoxicity on a wide variety of tumor cell lines in vitro (Laster, S. M. et al., J. Immunol. 141:2629-2634 (1988)). However, the mechanism of cell death induced by TNF has been largely unknown. HeLa cells express predominantly p55 TNF receptor which is thought to be responsible for cell death signaling (Englemann, H. et al., J. Biol. Chem. 265:14497-14504 (1990); Thoma, B. et al., J. Exp. Med. 172:1019-1023 (1990)). Additionally, HeLa cells are readily killed by TNF-xcex1 in the presence of the metabolic inhibitor cycloheximide (CHX). The cell death induced by TNF-xcex1/CHX shows DNA fragmentation and cytolysis, which are typical features of apoptosis (White, E. et al., Mol. Cell. Biol. 12:2570-2580 (1992)). Expression of adenovirus E1B 19K protein, which is functionally similar to bcl-2, inhibits apoptosis induced by TNF in HeLa cells (White, E. et al., Mol. Cell. Biol. 12:2570-2580 (1992)).
It has now been found that the IL-1xcex2 receptor antagonist (IL-1Ra) inhibits apoptosis induced by trophic factor deprivation and by hypoxia. In addition, mature IL-1xcex2 itself induces cell death through a pathway independent of CrmA-sensitive gene activity and cooperates with ICE and ICH-1L in apoptosis. As such, the invention identifies proIL-1xcex2 as the first substrate of any apoptosis inducing gene, whose cleavage product is a downstream mediator of the apoptotic cascade.
The invention is first directed to a method of preventing programmed cell death comprising the step of blocking mIL-xcex2 receptor binding. Preferably the mIL-xcex2 receptor binding is blocked with IL-1RA.
The invention is further directed to a method for inhibiting oncogenic transformation comprising stimulating apoptosis in infected cells. Preferably, the apoptosis is stimulated with IL-1xcex2 and/or TNF-xcex1.
The invention is further directed to a method of modulating apoptosis comprising activating the ICE pathway and mIL-1xcex2 production.
The invention is further directed to a method of modulating apoptosis comprising priming a cell prior to binding of IL-1 to its receptor. Priming the cell can include inter alia, use of trophic factor deprivation, hypoxia, G1/S phase arrest. This may be followed by IL-1xcex2 treatment.
The invention is further directed to a method of inhibiting hypoxia-inducted cell death using an IL-1 receptor blocker. Preferably the IL-1 receptor blocker is selected from the group consisting of IL-1Ra, an anti-IL-1polyclonal neutralizing antibody and an anti-IL-1 type-1 receptor neutralizing monoclonal antibody.
The invention is further directed to a method of preventing cell death resulting from ICH-1l comprising use of IL-1Ra.
Methods of use are provided. These include, inter alia, methods to either increase or decrease cell death in treating various pathologies, including tumors of specific bodily organs of an animal, including humans. Additionally, one may use the invention to inhibit oncogenic cell transformation, to address complications concerning apoptosis which accompany hypoxia or ischemia in various organs or to screen for agents which affect apoptosis.